Crosstalk between Smad and Mitogen-Activated Protein Kinases for the Regulation of Apoptosis in Cyclosporine A- Induced Renal Tubular Injury

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Measurement of intracellular generation of hydrogen peroxide by rat glomeruli in vitro

Sequential reduction of oxygen along the univalent pathway leads to the generation of superoxide anion, hydrogen peroxide, hydroxyl radical, and water [1, 2]. These partially reduced oxygen intermediates have been implicated as important mediators in various models of ischemic, toxic and immune-mediated tissue injury including glomerular injury [3]. Reactive oxygen metabolites have been shown to affect several biological processes potentially important in glomerular diseases, and their role in both inflammatory as well as non-inflammatory glomerular diseases has recently been demonstrated [3]. Evidence for the importance of reactive oxygen metabolites in experimental models of glomerular disease is based largely on the protective effects of scavengers of reactive oxygen metabolites

Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity

Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity.BackgroundIron plays a role in free radical-mediated tissue injury, including cisplatin-induced nephrotoxicity. However, the source of iron (catalyzing free radical reactions) is not known. We examined the role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity both in vivo and in vitro.MethodsCisplatin-induced acute renal failure was produced in rats by intraperitoneal injection of cisplatin (10mg/kg body wt). Piperonyl butoxide, a cytochrome P-450 inhibitor, was administered intraperitoneally (400mg/kg body wt twice at 48-hr intervals) prior to cisplatin injection. The effects of cisplatin in the absence or presence of piperonyl butoxide on the belomycin-detectable iron, cytochrome P-450 content in the kidney, and renal functional and histological changes were evaluated. In an in vitro study, the effect of cytochrome P-450 inhibitors, cimetidine or piperonyl butoxide, on cisplatin-induced cytotoxicity and catalytic iron release from LLC-PK1 cells was examined.ResultsIn cisplatin-treated rats, there was a marked decrease in the cytochrome P-450 content specifically in the kidney, accompanied by increased bleomycin-detectable iron content in the kidney. Piperonyl butoxide prevented cisplatin-induced loss of cytochrome P-450 as well as the increase of bleomycin-detectable iron in the kidney, along with both functional and histological protection. Both cimetidine and piperonyl butoxide prevented cisplatin-induced increase in bleomycin-detectable iron and cytotoxicity in LLC-PK1 cells. Treatment of cimetidine did not affect cellular uptake of cisplatin.ConclusionCytochrome P-450, a group of heme proteins, may serve as a significant source of catalytic iron in cisplatin-induced nephrotoxicity

In vitro and in vivo evidence suggesting a role for iron in cisplatin-induced nephrotoxicity

In vitro and in vivo evidence suggesting a role for iron in cisplatin-induced nephrotoxicity. Cisplatin is a widely used antineoplastic agent that has nephrotoxicity as a major side effect. The underlying mechanism of this nephrotoxicity is still not well known. Iron has been implicated to play an important role in several models of tissue injury, presumably through the generation of hydroxyl radicals via the Haber-Weiss reaction or other highly toxic free radicals. In the present study we examined the catalytic iron content and the effect of iron chelators in an in vitro model of cisplatin-induced cytotoxicity in LLC-PK1 cells (renal tubular epithelial cells) and in an in vivo model of cisplatin-induced acute renal failure in rats. Exposure of LLC-PK1 cells to cisplatin resulted in a significant increase in bleomycin-detectable iron (iron capable of catalyzing free radical reactions) released into the medium. Concurrent incubation of LLC-PK1 cells with iron chelators including deferoxamine and 1,10-phenanthroline significantly attenuated cisplatin-induced cytotoxicity as measured by lactate dehydrogenase (LDH) release. Bleomycin-detectable iron content was also markedly increased in the kidney of rats treated with cisplatin. Similarly, administration of deferoxamine in rats provided marked functional (as measured by blood urea nitrogen and creatinine) and histological protection against cisplatin-induced acute renal failure. In a separate study, we examined the role of hydroxyl radical in cisplatin-induced nephrotoxicity. Incubation of LLC-PK1 cells with cisplatin caused an increase in hydroxyl radical formation. Hydroxyl radical scavengers, dimethyl sulfoxide, mannitol and benzoic acid, significantly reduced cisplatin-induced cytotoxicity and, treatment with dimethyl sulfoxide or dimethylthiourea provided significant protection against cisplatin-induced acute renal failure. Taken together, our data strongly support a critical role for iron in mediating tissue injury via hydroxyl radical (or a similar oxidant) in this model of nephrotoxicity

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2. Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration ([Ca2+]i) in cultured LLC-PK1, cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10-7 M) increased [Ca2+]i from 148 ± 16 to 288 ± 39 nM and from 130 ± 8 to 240 ± 18 nM, with the values of EC50 for PAF and ONO11113 being 17 ± 4 and 17 ± 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO1113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(β-aminomethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). In the absence of extracellular Ca2+ 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (which has extensively been used to study the contribution of Ca2+ released from intracellular storage sites in the increase in [Ca2+]i), blocked the increment in [Ca2+]i induced by PAF or ONO1113. These results indicate that LLC-PKi cells express discrete receptors for PAF and TXA2 that are coupled to an increase in [Ca2+]i through mobilization of calcium from both intracellular storage sites and extracellular milieu, and suggest the possible importance of PAF and TXA2 in tubular pathophysiology

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury

Reactive oxygen species (ROS) modulate sphingolipid metabolism, including enzymes that generate ceramide and sphingosine-1-phosphate (S1P), and a ROS-antioxidant rheostat determines the metabolism of ceramide-S1P. ROS induce ceramide production by activating ceramide-producing enzymes, leading to apoptosis, while they inhibit S1P production, which promotes survival by suppressing sphingosine kinases (SphKs). A ceramide-S1P rheostat regulates ROS-induced mitochondrial dysfunction, apoptotic/anti-apoptotic Bcl-2 family proteins and signaling pathways, leading to apoptosis, survival, cell proliferation, inflammation and fibrosis in the kidney. Ceramide inhibits the mitochondrial respiration chain and induces ceramide channel formation and the closure of voltage-dependent anion channels, leading to mitochondrial dysfunction, altered Bcl-2 family protein expression, ROS generation and disturbed calcium homeostasis. This activates ceramide-induced signaling pathways, leading to apoptosis. These events are mitigated by S1P/S1P receptors (S1PRs) that restore mitochondrial function and activate signaling pathways. SphK1 promotes survival and cell proliferation and inhibits inflammation, while SphK2 has the opposite effect. However, both SphK1 and SphK2 promote fibrosis. Thus, a ceramide-SphKs/S1P rheostat modulates oxidant-induced kidney injury by affecting mitochondrial function, ROS production, Bcl-2 family proteins, calcium homeostasis and their downstream signaling pathways. This review will summarize the current evidence for a role of interaction between ROS-antioxidants and ceramide-SphKs/S1P and of a ceramide-SphKs/S1P rheostat in the regulation of oxidative stress-mediated kidney diseases

Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed

Impact of Inflammation on Ferritin, Hepcidin and the Management of Iron Deficiency Anemia in Chronic Kidney Disease

Iron deficiency anemia (IDA) is a major problem in chronic kidney disease (CKD), causing increased mortality. Ferritin stores iron, representing iron status. Hepcidin binds to ferroportin, thereby inhibiting iron absorption/efflux. Inflammation in CKD increases ferritin and hepcidin independent of iron status, which reduce iron availability. While intravenous iron therapy (IIT) is superior to oral iron therapy (OIT) in CKD patients with inflammation, OIT is as effective as IIT in those without. Inflammation reduces predictive values of ferritin and hepcidin for iron status and responsiveness to iron therapy. Upper limit of ferritin to predict iron overload is higher in CKD patients with inflammation than in those without. However, magnetic resonance imaging studies show lower cutoff levels of serum ferritin to predict iron overload in dialysis patients with apparent inflammation than upper limit of ferritin proposed by international guidelines. Compared to CKD patients with inflammation, optimal ferritin levels for IDA are lower in those without, requiring reduced iron dose and leading to decreased mortality. The management of IDA should differ between CKD patients with and without inflammation and include minimization of inflammation. Further studies are needed to determine the impact of inflammation on ferritin, hepcidin and therapeutic strategy for IDA in CKD

Mycoplasma pneumoniae-associated mild encephalitis/encephalopathy with a reversible splenial lesion: report of two pediatric cases and a comprehensive literature review

Abstract Background No literature review exists on Mycoplasma pneumoniae-associated mild encephalitis/encepharopathy with a reversible splenial lesion (MERS). Methods M.pneumoniae-associated MERS cases were searched till August 2016 using PubMed/Google for English/other-language publications and Ichushi ( http://www.jamas.or.jp/ ) for Japanese-language publications. Inclusion criteria were children fulfilling definition for encephalitis, M.pneumoniae infection, and neuroimaging showing hyperintensity in the splenium of the corpus callosum (SCC) alone (type I) or SCC/other brain areas (type II). Results We described two children with type I and II M.pneumoniae-associated MERS. Thirteen cases found by the search and our 2 cases were reviewed. Mean age, male/female ratio, duration of prodromal illness was 8.3 years, 1.5 and 3.5 days. The most common neurological symptom was drowsiness, followed by abnormal speech/behavior, ataxia, seizure, delirium, confusion, tremor, hallucination, irritability, muscle weakness, and facial nerve paralysis. Fever was the most common non-neurological symptom, followed by cough, headache, gastrointestinal symptoms, headache, lethargy and dizziness. Seizure and respiratory symptoms were less common. All were diagnosed for M.pneumoniae by serology. Cerebrospinal fluid (CSF) M.pneumoniae was undetectable by PCR in the 3 patients. Three patients were clarithromycin-resistant. Leukocytosis, positive C-reactive protein, hyponatremia, CSF pleocytosis and slow wave on electroencephalography frequently occurred. All except 2 were type I MERS. Neuroimaging abnormalities disappeared within 18 days in the majority of patients. All type I patients completely recovered within 19 days. Two type II patients developed neurological sequelae, which recovered 2 and 6 months after onset. Conclusions Prognosis of M.pneumoniae-associated MERS is excellent. Type II MERS may increase a risk of neurological sequelae

Hepcidin-25, Mean Corpuscular Volume, and Ferritin as Predictors of Response to Oral Iron Supplementation in Hemodialysis Patients

The benefit of oral iron therapy (OIT) and factors predictive of OIT response are not established in hemodialysis (HD) patients with iron deficiency anemia (IDA). We examined the values of hepcidin-25, mean corpuscular volume (MCV), and ferritin as predictors of OIT response. Oral ferrous fumarate (50 mg/day, 8 weeks) was given to 51 HD patients with IDA (hemoglobin (Hb) < 12 g/dL, ferritin < 100 ng/mL) treated with an erythropoietin activator. Sixteen patients were responders (improvement of Hb (ΔHb) ≥ 2 g/dL) and 35 were non-responders (ΔHb < 2g/dL). Baseline Hb, MCV, serum hepcidin-25, ferritin, iron parameters, and C-reactive protein (CRP) before and ΔHb after OIT were compared between groups. Hepcidin-25, MCV, ferritin, and transferrin saturation were lower in the responders than in the non-responders. Hepcidin-25 positively correlated with ferritin. Hepcidin-25, MCV, and ferritin positively correlated with baseline Hb and negatively correlated with ΔHb. Despite normal CRP levels in all patients, CRP correlated positively with hepcidin-25 and ferritin. Stepwise multiple linear regression analysis and receiver operating characteristics curve analysis revealed that hepcidin-25, MCV, and ferritin could predict OIT response. We conclude that hepcidin-25, MCV, and ferritin could be useful markers of iron storage status and may help predict OIT response in HD patients